The term "Listeria" as used herein, refers to the bacteria classified as such in Bergey's Manual of Systematic Bacteriology (P. H. A. Sneath (ed), 1986, 1234-1245, Williams & Wilkins). Recent 16S rRNA sequence data of Listeria murrayi and Listeria grayi together with well known phenotypic uniformities have led to a recent proposal to retain these two groups in the Listeria genus (Rocourt et al., International Journal of Systematic Bacteriology, Vol. 37, No. 3, pp 298-300 (1987)). Therefore, the term "Listeria" as used herein includes Listeria monocytogenes, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria ivanovii, Listeria murrayi and Listeria grayi.
Detection of Listeria is important in various medical and public health contexts. Human listeriosis has been shown to be clearly related to consumption of Listeria-contaminated foods only in the past few years. The outbreaks documenting this association (Schlech et al., New England J. Med. 308: 203-206 (1983); Fleming et al., New Eng. J. Med. 312: 404-407 (1985); U.S. Public Health Service, Centers for Disease Control, Morb. Mortal. Rpt. 34: 357-359 (1985)) have caused concern among public health officials, food manufacturers and regulatory agencies. Prior to these outbreaks, food microbiologists have been generally unaware and uninformed about this nearly ubiquitous group of organisms and the associated disease manifestations. Most efforts to improve diagnostic methods for Listeria have been based on its occurrence as a veterinary pathogen or as a cause of human neonatal infection (Hird, J. Food Protection 50: 429-433 (1987)).
Many samples cultured for Listeria (including foods) contain large, mixed microbial populations which hamper recovery and identification. Therefore, it is an aspect of the present invention to provide a novel assay system capable of rapidly detecting Listeria and which is generally applicable to environmental, food or clinical samples.
Because Listeria species other than L. monocytogenes have been recovered from a variety of food types, detection of such organisms in a food-processing environment may be important by virtue of their "indicator organism" status.
The presence of Listeria has been historically detected by culturing an appropriately prepared sample on microbiological media under conditions favorable for growth of these organisms (Lovett et al., J. Food Protection 50: 188-192 [1987], Anonymous, Fed. Register 53: 44148-44153 [1988]). McClain and Lee, J. Assoc. Off. Anal. Chem., 71: 660-664 [1988]). The resulting colonies are typically examined for morphological and biochemical characteristics, a process that generally is initiated 48 hours after acquisition of the sample and disadvantageously takes between 4-5 days to complete.
It is another aspect of the present invention to avoid the disadvantage associated with traditional, multi-day culturing techniques and to employ nucleic acid probes to detect Listeria.
It is yet another aspect of the present invention to provide probes which can hybridize to target regions which can be rendered accessible to the probes under normal assay conditions.
While Kohne et al., Biophysical Journal 8: 1104-1118 (1968), discuss one method for preparing probes to rRNA sequences they do not provide the teaching necessary to make Listeria specific probes.
U.S. Pat. No. 5,089,386 of Stackebrandt et al., filed Sep. 11, 1987 describes Listeria specific probes and while such probes work well, it is another aspect of the present invention to provide novel and improved Listeria probes not disclosed therein.
Pace and Campbell, Journal of Bacteriology 107: 543-547 (1971), discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. Similarly, Sogin, Sogin, and Woese, Journal of Molecular Evolution 1: 173-184 (1972), discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships. Fox, Pechman, and Woese, International Journal of Systematic Bacteriology (1977), discuss the comparative cataloging of 16S ribosomal RNAs as an approach to prokaryotic systematics. These references, however, fail to relieve the deficiency of Kohne's teaching with respect to Listeria and in particular, do not provide Listeria specific probes useful in assays for detecting Listeria in food and other samples.
Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Ribosomes contain three distinct RNA molecules which, at least in E. coli, are referred to as 5S, 16S, and 23S rRNAs. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosome molecules vary substantially in size between organisms.
Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacteria, and this convention will be continued herein.
Hybridization traditionally is understood as the process by which, under predetermined reaction conditions, two partially or completely complementary single-stranded nucleic acids are allowed to come together in an antiparallel fashion to form a double-stranded nucleic acid with specific and stable hydrogen bonds.
The stringency of a particular set of hybridization conditions is defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids.
Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and/or the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will largely dictate the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art.
As a general matter, dependent upon probe length, such persons understand stringent conditions to mean approximately 35.degree. C.-65.degree. C. in a salt solution of approximately 0.9 molar.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially) to target nucleic acid sequences. Two functionally modified probes are described in the examples described herein.
Capture probes are modified in such a way that they hybridize to the target nucleic acid molecule and can be removed, along with the target molecule, from solution. Detection probes are advantageously modified by the addition of one of a variety of detectable ligands (e.g. .sup.32 P, biotin, fluorescein, etc.) which permit the direct or indirect detection of the target nucleic acid.
A target nucleic acid sequence is one to which a particular probe is capable of preferentially hybridizing.
Still other useful definitions are given as their first use arises in the following text. All references cited herein are fully incorporated by reference.